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Ecotoxicology and Environmental Safety Jun 2024Brassinosteroids (BRs) can regulate various processes in plant development and defense against environmental stress. In this study, the contribution of BRs in the...
Brassinosteroids (BRs) can regulate various processes in plant development and defense against environmental stress. In this study, the contribution of BRs in the degradation of isoproturon (IPU) in rice has been established. IPU has a significant effect on rice growth, chlorophyll content, and membrane permeability. When treated with 1.0 μmol/L 24-epibrassinolide (EBR), a BR analogue, the associated symptoms of rice poisoning were alleviated as the IPU levels in the rice and growth media were decreased. In the presence of EBR, the activities of several IPU-related detoxification enzymes were enhanced to cope with the stress due to IPU. An RNA-sequencing (RNA-Seq) has been performed to determine the variation of transcriptomes and metabolic mechanisms in rice treated with EBR, IPU, or IPU+EBR. Some of the differentially expressed genes (DEGs) were Phase I-III reaction components of plants, such as cytochrome P450 (CYP450), glutathione S-transferase (GST), glycosyltransferases (GTs), and the ATP-binding cassette transporter (ABC transporter). The expression of some signal transduction genes was significantly up-regulated. The relative content of low-toxicity IPU metabolites increased due to the presence of EBR as determined by UPLC/Q-TOF-MS/MS. The IPU metabolic pathways include enzyme-catalyzed demethylation, hydroxylation, hydrolysis, glycosylation, and amino acid conjugation processes. The results suggest that EBR plays a key role in the degradation and detoxification of IPU. This study has provided evidence that BRs regulate the metabolism and detoxification of IPU in rice, and offers a new approach to ensuring cleaner crops by eliminating pesticide residues in the environment.
PubMed: 38917591
DOI: 10.1016/j.ecoenv.2024.116575 -
Journal of the American Chemical Society Jun 2024The receptor for advanced glycation end products (RAGE) plays a crucial role in inflammation-related pathways and various chronic diseases. Despite the recognized...
The receptor for advanced glycation end products (RAGE) plays a crucial role in inflammation-related pathways and various chronic diseases. Despite the recognized significance of N-glycosylation in the ligand-binding V domain (VD) of RAGE, a comprehensive understanding of the site-activity and structure-activity relationships is lacking due to the challenges in obtaining homogeneous glycoprotein samples through biological expression. Here, we combined chemical and chemoenzymatic approaches to synthesize RAGE-VD and its congeners with Asn3-glycosylation by incorporating precise N-glycan structures. Evaluation of these samples revealed that, in comparison to other RAGE-VD forms, α2,6-sialylated N-glycosylation at the Asn3 site results in more potent inhibition of HMGB1-induced nuclear factor-κB (NF-κB) expression in RAGE-overexpressing cells. Hydrogen/deuterium exchange-mass spectrum analysis revealed a sialylated RAGE-VD-induced interaction region within HMGB1. Conversely, Asn3 N-glycosylation in VD has negligible effects on RAGE-VD/S100B interactions. This study established an approach for accessing homogeneously glycosylated RAGE-VD and explored the modulatory effects of N-glycosylation on the interactions between RAGE-VD and its ligand proteins.
PubMed: 38917169
DOI: 10.1021/jacs.4c01413 -
Proceedings of the National Academy of... Jul 2024The fucosylation of glycoproteins regulates diverse physiological processes. Inhibitors that can control cellular levels of protein fucosylation have consequently...
The fucosylation of glycoproteins regulates diverse physiological processes. Inhibitors that can control cellular levels of protein fucosylation have consequently emerged as being of high interest. One area where inhibitors of fucosylation have gained significant attention is in the production of afucosylated antibodies, which exhibit superior antibody-dependent cell cytotoxicity as compared to their fucosylated counterparts. Here, we describe β-carbafucose, a fucose derivative in which the endocyclic ring oxygen is replaced by a methylene group, and show that it acts as a potent metabolic inhibitor within cells to antagonize protein fucosylation. β-carbafucose is assimilated by the fucose salvage pathway to form GDP-carbafucose which, due to its being unable to form the oxocarbenium ion-like transition states used by fucosyltransferases, is an incompetent substrate for these enzymes. β-carbafucose treatment of a CHO cell line used for high-level production of the therapeutic antibody Herceptin leads to dose-dependent reductions in core fucosylation without affecting cell growth or antibody production. Mass spectrometry analyses of the intact antibody and -glycans show that β-carbafucose is not incorporated into the antibody -glycans at detectable levels. We expect that β-carbafucose will serve as a useful research tool for the community and may find immediate application for the rapid production of afucosylated antibodies for therapeutic purposes.
Topics: Fucose; Animals; CHO Cells; Cricetulus; Glycosylation; Humans; Trastuzumab; Fucosyltransferases; Antibody-Dependent Cell Cytotoxicity
PubMed: 38917011
DOI: 10.1073/pnas.2314026121 -
Frontiers in Immunology 2024This study aimed to investigate the dynamics of programmed death-ligand 1 (PD-L1) expression, spatial heterogeneity, and binding affinity of FDA-approved anti-PD-L1...
INTRODUCTION
This study aimed to investigate the dynamics of programmed death-ligand 1 (PD-L1) expression, spatial heterogeneity, and binding affinity of FDA-approved anti-PD-L1 antibodies (avelumab and atezolizumab) in gastric cancer. Additionally, we determined how PD-L1 glycosylation impacts antibody accumulation in gastric cancer cells.
METHODS
Dynamic PD-L1 expression was examined in NCIN87 gastric cancer cells. Comparative binding studies of avelumab and atezolizumab were conducted in gastric cancer models, both and . Antibody uptake in tumors was visualized through positron emission tomography (PET) imaging. PD-L1 glycosylation status was determined via Western blot analyses before and after PNGase F treatment.
RESULTS
Consistent findings revealed time-dependent PD-L1 induction in NCIN87 gastric cancer cells and spatial heterogeneity in tumors, as shown by PET imaging and immunofluorescence. Avelumab displayed superior binding affinity to NCIN87 cells compared to atezolizumab, confirmed by PET imaging and biodistribution analyses. Notably, PD-L1 glycosylation at approximately 50 kDa was observed, with PNGase F treatment inducing a shift to 35 kDa in molecular weight. Tissue samples from patient-derived xenografts (PDXs) validated the presence of both glycosylated and deglycosylated PD-L1 (degPD-L1) forms in gastric cancer. Immunofluorescence microscopy and binding assays demonstrated enhanced avelumab binding post-deglycosylation.
DISCUSSION
This study provides an understanding of dynamic and spatially heterogeneous PD-L1 expression in gastric cancer. Anti-PD-L1 immunoPET was able to visualize gastric tumors, and PD-L1 glycosylation has significant implications for antibody recognition. These insights contribute to demonstrating the complexities of PD-L1 in gastric cancer, holding relevance for refining PD-L1 imaging-based approaches.
Topics: Stomach Neoplasms; B7-H1 Antigen; Humans; Animals; Mice; Cell Line, Tumor; Glycosylation; Antibodies, Monoclonal, Humanized; Xenograft Model Antitumor Assays; Female; Positron-Emission Tomography
PubMed: 38915392
DOI: 10.3389/fimmu.2024.1405485 -
Acta Biochimica Et Biophysica Sinica Jun 2024FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase expressed in hematopoietic cells. Internal-tandem duplication domain (ITD) mutation and tyrosine kinase...
FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase expressed in hematopoietic cells. Internal-tandem duplication domain (ITD) mutation and tyrosine kinase domain (TKD) mutation are the two most common mutations in acute myeloid leukemia (AML). Post-translational modifications (PTMs) of FLT3, such as glycosylation and ubiquitination, have been shown to impact various aspects of the protein in both wild-type (WT) and mutant forms of FLT3. In this review, we describe how the glycosylation status of FLT3 affects its subcellular localization, which significantly impacts the activation of downstream signaling, and the impact of specific ubiquitination on FLT3 function and stability, which may be associated with disease progression. Moreover, potential novel therapeutic strategies involving a combination of FLT3 tyrosine kinase inhibitors and drugs targeting glycosylation or ubiquitination are discussed.
PubMed: 38915288
DOI: 10.3724/abbs.2024112 -
[Analysis of enzyme activity and substrate specificity of dolichyl-phosphate β-glucosyltransferase].Sheng Wu Gong Cheng Xue Bao = Chinese... Jun 2024Protein folding and quality control processes primarily occur in the endoplasmic reticulum (ER). ER-resident molecular chaperones play a crucial role in guiding nascent...
Protein folding and quality control processes primarily occur in the endoplasmic reticulum (ER). ER-resident molecular chaperones play a crucial role in guiding nascent polypeptides towards their correct tertiary structures. Some of these chaperones specifically recognize glucosylated -glycan moieties on peptide. It is of great significance to study the -glycan biosynthetic pathway and glycoprotein quality control system by analyzing the sugar donor of ER luminal glucosyltransferases, known as dolichol phosphate glucose (Dol-P-Glc), or its analogues . In this study, we investigated a range of dolichol analogues to synthesize lipid phosphate glucose, which served as substrates for dolichyl-phosphate β-glucosyltransferase E (Alg5E) derived from . The results demonstrated that the recombinant Alg5E, expressed in , exhibited strong catalytic activity and the ability to recognize lipid phosphate glucose with varying chain lengths. Interestingly, the enzyme's catalytic reaction was found to be faster with longer carbon chains in the substrate. Additionally, Alg5E showed a preference for branched chain methyl groups in the lipid structure. Furthermore, our study confirmed the importance of divalent metal ions in the binding of the crucial DXD motif, which is essential for the enzyme's catalytic function. These findings lay the groundwork for future research on glucosyltransferases Alg6, Alg8, and Alg10 in the synthesis pathway of dolichol-linked oligosaccharide (DLO).
Topics: Glucosyltransferases; Substrate Specificity; Escherichia coli; Trichomonas vaginalis; Recombinant Proteins; Dolichol Phosphates; Endoplasmic Reticulum
PubMed: 38914494
DOI: 10.13345/j.cjb.230737 -
Neuroscience Letters Jun 2024Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) is a pleiotropic peptide known to promote many beneficial processes following neural damage and cell death...
Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) is a pleiotropic peptide known to promote many beneficial processes following neural damage and cell death after stroke. Despite PACAP's known neurotrophic and anti-inflammatory properties, it has not realized its translational potential due to a poor pharmacokinetic profile (non-linear PK/PD), and limited Blood-Brain Barrier Penetration (BBB) permeability. We have previously shown that glycosylation of PACAP increases stability and enhances BBB penetration. In addition, our prior studies showed reduced neuronal cell death and neuroinflammation in models of Parkinson's disease and Traumatic Brain Injury (TBI). In this study we show that a PACAP glucoside retains the known neurotrophic activity of native PACAPin vitro and a 5-day daily treatment regimen (100 nM) leads to neurite-like extensions in PC12 cells. In addition, we show that intraperitoneal injection of a PACAP lactoside (10 mg/kg) with improved BBB-penetration, given 1-hour after reperfusion in a Transient Middle Cerebral Artery Occlusion (tMCAO) mouse model, reduces the infarct size after the ischemic injury in males significantly by ∼ 36 %, and the data suggest a dose-dependency. In conclusion, our data support further development of PACAP glycopeptides as promising novel drug candidates for the treatment of stroke, an area with an urgent clinical need.
PubMed: 38914278
DOI: 10.1016/j.neulet.2024.137883 -
Journal of Agricultural and Food... Jun 2024Lipase from (ROL) exhibits remarkable -1,3 stereoselectivity and catalytic activity, but its poor thermostability limits its applications in the production of...
Lipase from (ROL) exhibits remarkable -1,3 stereoselectivity and catalytic activity, but its poor thermostability limits its applications in the production of 1,3-dioleoyl-2-palmitoyl glycerol (OPO, a high-quality substitute for human milk fat). In this work, a semirational method was proposed to engineer the thermostability and catalytic activity of 4M (ROL mutant in our previous study). First, a computer-aided design is performed using 4M as a template, and -glycosylation mutants are then recombinantly expressed and screened in , the optimal mutant N227 exhibited a half-life of 298.8 h at 45 °C, which is 7.23-folds longer than that of 4M. Its catalytic activity also reached 1043.80 ± 61.98 U/mg, representing a 29.2% increase compared to 4M (808.02 ± 47.02 U/mg). Molecular dynamics simulations of N227 suggested that the introduction of glycan enhanced the protein rigidity, while the strong hydrogen bonds formed between the glycan and the protein stabilized the lipase structure, thereby improving its thermostability. The acidolysis reaction between oleic acid (OA) and glycerol tripalmitate (PPP) was successfully carried out using immobilized N227, achieving a molar conversion rate of 90.2% for PPP. This engineering strategy guides the modification of lipases, while the glycomutants obtained in this study have potential applications in the biosynthesis of OPO.
PubMed: 38913033
DOI: 10.1021/acs.jafc.4c02623 -
JCI Insight May 2024The regulated glycosylation of the proteome has widespread effects on biological processes that cancer cells can exploit. Expression of N-acetylglucosaminyltransferase V...
The regulated glycosylation of the proteome has widespread effects on biological processes that cancer cells can exploit. Expression of N-acetylglucosaminyltransferase V (encoded by Mgat5 or GnT-V), which catalyzes the addition of β1,6-linked N-acetylglucosamine to form complex N-glycans, has been linked to tumor growth and metastasis across tumor types. Using a panel of murine pancreatic ductal adenocarcinoma (PDAC) clonal cell lines that recapitulate the immune heterogeneity of PDAC, we found that Mgat5 is required for tumor growth in vivo but not in vitro. Loss of Mgat5 results in tumor clearance that is dependent on T cells and dendritic cells, with NK cells playing an early role. Analysis of extrinsic cell death pathways revealed Mgat5-deficient cells have increased sensitivity to cell death mediated by the TNF superfamily, a property that was shared with other non-PDAC Mgat5-deficient cell lines. Finally, Mgat5 knockout in an immunotherapy-resistant PDAC line significantly decreased tumor growth and increased survival upon immune checkpoint blockade. These findings demonstrate a role for N-glycosylation in regulating the sensitivity of cancer cells to T cell killing through classical cell death pathways.
Topics: Animals; Glycosylation; Mice; N-Acetylglucosaminyltransferases; Carcinoma, Pancreatic Ductal; Pancreatic Neoplasms; Cell Line, Tumor; Humans; T-Lymphocytes; Dendritic Cells; Killer Cells, Natural; Mice, Knockout
PubMed: 38912584
DOI: 10.1172/jci.insight.178804 -
Acta Biochimica Et Biophysica Sinica Jun 2024N-glycans play important roles in a variety of biological processes. In recent years, analytical technologies with high resolution and sensitivity have advanced...
N-glycans play important roles in a variety of biological processes. In recent years, analytical technologies with high resolution and sensitivity have advanced exponentially, enabling analysts to investigate N-glycomic changes in different states. Specific glycan and glycosylation signatures have been identified in multiple diseases, including cancer, autoimmune diseases, nervous system disorders, and metabolic and cardiovascular diseases. These glycans demonstrate comparable or superior indicating capability in disease diagnosis and prognosis over routine biomarkers. Moreover, synchronous glycan alterations concurrent with disease initiation and progression provide novel insights into pathogenetic mechanisms and potential treatment targets. This review elucidates the biological significance of N-glycans, compares the existing glycomic technologies, and delineates the clinical performance of N-glycans across a range of diseases.
PubMed: 38910518
DOI: 10.3724/abbs.2024101